The availability of nucleic acid sequence information has enabled large-scale and detailed analysis of cellular and molecular processes. Among the many applications for such analyses, it is convenient to attach a nucleic acid to a solid support.
Arrays of nucleic acids are used to locate different nucleic acid sequences in an addressable format. The arrays have applications in analyzing nucleic acids in a sample, analyzing genetic polymorphisms, nucleic acid sequencing, research, and diagnostics. By design, the arrays are suited for high-throughput analysis.
For example, nucleic acid arrays can be used for the for large-scale gene expression studies (see, e.g., U.S. Pat. No. 5,889,165). These studies determine the levels of expression of a large proportion of genes present in the genome. Generally, the arrays can be used in a variety of hybridization assays, but also in enzymatic reactions such as nucleic acid extension reactions. For example, nucleic acid arrays can be used for solid phase PCR (see, e.g., WO 01/48242 A2), sequencing by hybridization (see, e.g., U.S. Pat. No. 5,695,940), and single-base extension reactions (see, e.g., U.S. Pat. No. 6,004,774).
A variety of chemistries can be used to link a nucleic acid to a solid support. In one method, the nucleic acid is modified with a sulfhydryl group and reacted with a mercaptosilane coated solid phase. See, e.g., U.S. Pat. No. 6,030,782. U.S. Pat. No. 6,169,194 describes high-density immobilization of sulfhydryl modified oligonucleotides using a permanent thioether bond. In still another method, a covalent bond between a modified oligonucleotide and a solid phase surface is formed by a condensation reaction with a water-soluble carbodiimide as described by Rasmussen et al. (1991) Anal. Biochem. 198:138–142. Markos et al. (1992) Nucl. Acids Res. 20:1679–1684 describes a flexible linker with a primary hydroxyl group that is coupled to the solid phase by a glycidoxypropyl silane. U.S. Pat. No. 6,171,797 describes a cycloaddition reaction between a diene with a dienophile to couple a nucleotide to a substrate.
Another method of attaching nucleic acids to solid supports uses amine-modified nucleic acids. The amine group is used as a functional group that reacts with a solid support. For example, the solid support can be an aldehyde coated glass substrate (see, e.g., U.S. Pat. No. 6,030,782). The amine linkage is robust and can be used to orient the nucleic acid with respect the solid support by selective introduction of the amine group.
One method of synthesizing amino-modified nucleic acids uses β-cyanoethyl phosphoramidites that are attached to the 5′ terminus of an oligonucleotide.